The objective of this study is to identify and characterize the viral proteins and RNA sequences that are directly involved in poliovirus (+) and (-) strand RNA synthesis in vivo. In preliminary studies, we have shown that transcripts of the clone pT7D-polio are infectious and that they replicate in transfected cells. A subgenomic RNA with in-frame deletions in the capsid coding region was self-replicating, whereas a mutant RNA with an out-of-frame deletion in the same site did not replicate either alone or in the presence of helper RNA. Thus, one or more of the replication proteins must contain a cis active function. A mutant RNA with a Pl-2A deletion synthesized small amounts of RNA at late times posttransfection. Cotransfection with helper RNA dramatically increased the replication this mutant RNA. Thus, it was possible to complement this 2A mutant in trans using the cotransfection procedure. In this study, we will characterize additional RNA-negative mutants and conduct complementation studies to identify sequences encoding cis and trans active functions. The ability to produce smaller subgenomic replicons will be investigated by deleting sequences encoding trans active functions. To determine if the viral replication proteins can be supplied in trans for the replication of (-) strand RNA, we will determine if (+) strand RNA can be synthesized (-) strand templates in the presence of helper RNA. A final objective will be the identification of RNA sequences and/or structures in the 3' terminal ends of (+) and (-) strand viral RNAs that are directly required for RNA replication. The results of this study will provide us with important new insights into the unique genetic and replication strategies that are used by poliovirus and perhaps other (+) strand RNA viruses.